Biomineralization is one of the most widespread and important processes in both vertebrates and invertebrates. We have directed our efforts to study the mineralization of bone and dentin in which apatite crystals are specifically grown within type I collagen matrices. Although it is likely that the overall mechanisms involved in mineralization of the two tissues are similar, we have adopted the dentin system since it represents a relatively simpler system than bone. The basic working hypothesis has been that certain acidic, phosphorylated, non-collagenous extracellular matrix protein macromolecules (NCP) are first localized by interaction with the collagen I fibril surfaces, which then direct the nucleation of crystal deposits and orientation of certain specific crystallographic habit directions relative to the fibril axes. Further interactions of the NCP with the growing crystals are postulated to specifically regulate crystal growth in terms of habit, shape and size. Although there are many NCP present in dentin, we have focused our efforts on the primary phosphoprotein of dentin: Phosphophoryn ([PP]). PP is very acidic in nature and characterized by a very high content of serine residues, at about an 85 percent level of phosphorylation. In this proposal we plan to introduce the gene encoding for PP to over-express it in fibroblast, osteoblast and odontoblast cells, respectively. This will enable us to establish an in vivo system to study biomineralization. We will characterize the mineralization of the crystals in these three separate cells to ascertain the nucleation and crystal growth directions. This approach will allow us to address many important questions: Is the post-translationnal modification of PP similar in fibroblast in contrast to osteoblast and/or odontoblast cells? Will the mineralized crystal have the same crystallographic properties when the nucleating protein, such as PP, is expressed in a fibroblast without the rest of the NCP, which is usually expressed in an osteoblast or odontoblast? We will also study the effect of phosphorylation on the Mineralization process in vitro by expressing the recombinant PP and studying its in vitro phosphorylation on the mineral. These studies should yield new information related to the specific role of phosphoproteins in biomineralization. This knowledge will be extremely valuable in determining the mechanisms involved in the biomineralization process particularly since the studies are conducted in an in vivo model in comparison to the traditional approaches of in vitro studies of biomineralization. This data will also provide basic knowledge for designing treatment of ectopic mineralization specifically in diseases as such kidney stones or failing heart valve prosthesis.